Abstract

Loss of presenilin function in adult mouse brains causes memory loss and age-related neurodegeneration. Since presenilin possesses gamma-secretase-dependent and -independent activities, it remains unknown which activity is required for presenilin-dependent memory formation and neuronal survival. To address this question, we generated postnatal forebrain-specific nicastrin conditional knock-out (cKO) mice, in which nicastrin, a subunit of gamma-secretase, is inactivated selectively in mature excitatory neurons of the cerebral cortex. nicastrin cKO mice display progressive impairment in learning and memory and exhibit age-dependent cortical neuronal loss, accompanied by astrocytosis, microgliosis, and hyperphosphorylation of the microtubule-associated protein Tau. The neurodegeneration observed in nicastrin cKO mice likely occurs via apoptosis, as evidenced by increased numbers of apoptotic neurons. These findings demonstrate an essential role of nicastrin in the execution of learning and memory and the maintenance of neuronal survival in the brain and suggest that presenilin functions in memory and neuronal survival via its role as a gamma-secretase subunit.

Levels of γsecretase subunits and APP cleaved products in nicastrin cKO and PS cDKO mice at 2 months of age

(A) Significant reductions of the levels of PS1-CTF, PS1-NTF, and Pen-2 but unchanged levels of Aph-1a in nicastrin cKO mice. Cortical lysates from nicastrin cKO and control mice were analyzed by immunoblotting (top) using antibodies against PS1-CTF, PS1-NTF, Pen-2 and Aph-1a. Protein levels were standardized with β-actin (bottom). The asterisk denotes statistical significance (*, p < 0.05). NS denotes not significant.(B) Reductions of γ secretase subunits in PS cDKO mice. Conditional removal of PSs results in an about 50% reduction of PS1-CTF and PS1-NTF levels, that is similar to the observation in nicastrin cKO. Reduced levels of Pen-2 but unchanged levels of Aph-1a are observed in PS cDKO mice. Cortical lysates from PS cDKO and control mice were analyzed by immunoblotting (top). Protein levels were standardized with β-actin (bottom). The asterisk denotes statistical significance (*, p < 0.05).(C, D) Unchanged levels of APP full-length (APP-FL) and soluble APP-α fragment (α APPs) in nicastrin cKO (C) and PS cDKO mice (D). Immunoblotting (top) was conducted using antibodies specific for APP-FL and α APPs. Protein levels were standardized with β-actin (bottom). Quantification analysis shows no significant changes in protein levels for APP-FL and α APPs, but a massive increase in protein levels for APP-CTFs in nicastrin cKO and PS cDKO mice, relative to those in control mice.(E) Direct comparison of immunoblotting on APP-CTFs in nicastrin cKO and PS cDKO mice at 2 months of age. Cortical lysates prepared from nicastrin cKO and PS cDKO mice were diluted as indicated (lanes 3–8), and immunoblotted with the APP C8 antibody. Undiluted homogenate (1X) and 4 times of the undiluted homogenate (4X) from control mice were also immunoblotted. The levels of APP-CTFs in nicastrin cKO and PS cDKO cortex were increased about 60-fold relative to those of the control mice.

(A) Normal brain cytoarchitecture, including the neocortex and hippocampus, revealed by Nissl staining of sagittal sections from nicastrin cKO and control brains.(B) Comparable MAP2 immunoreactivity in the neocortex and hippocampal area CA1 of cKO and control brains.(C) Comparable synaptophysin (SVP38) immunoreactivity in the neocortex and hippocampal area CA1 of cKO and control brains. Scale bar: 100 μm.

Progressive impairment of learning and memory in nicastrin cKO mice. Nicastrin cKO and control mice at two different ages, 2–3 and 7–8 months, were examined in the Morris watermaze to test spatial learning and memory, and in the contextual fear conditioning to test associative memory

(A) Escape latency in nicastrin cKO mice at 2–3 months. Nicastrin cKO mice display longer escape latencies than age-matched littermate control animals across the 7-day training period (n = 10 for control; n = 14 for nicastrin cKO).(B) Escape latency in nicastrin cKO mice at 7–8 months. Almost no improvement in escape latencies was observed in Nicastrin cKO mice across the 7-day training period (n = 15 for control; n = 11 for nicastrin cKO).(C) Probe trial in nicasrtrin cKO mice at 2–3 months of age. 2–3 months old nicastrin cKO mice spend significantly less time searching for the platform in the target quadrant than the controls. The asterisk denotes statistical significance (*, p < 0.05). AL: adjacent left quadrant; T: target quadrant; AR: adjacent right quadrant; OP: opposite quadrant. 25% is the chance performance during the probe trial.(D) Probe trial in nicastrin cKO mice at 7–8 months of age. The time spent in the target quadrant is dramatically decreased in 7–8 months old nicastrin cKO mice compared to the controls (F = 13.2, df1/24, p < 0.001). Of note, the time spent in the opposite quadrant where they were initially released is significantly increased in the cKO group (F = 12.9, df1/24, p < 0.001). The asterisk denotes statistical significance (***, p < 0.001).(E, F) Representative swim-paths for mice at 2–3 (E) and 7–8 (F) months of age during the post-training probe trial. TQ: target quadrant.(G) Percentage of time spent in freezing in nicastrin cKO mice at 2–3 months of age in the contextual fear conditioning. Nicastrin cKO mice spend significantly less time freezing than age-matched littermate control animals 24 hours after the footshock (p < 0.01; n = 28 for control; n = 25 for nicastrin cKO), however, they show indistinguishable levels of freezing to control mice during the training period. The asterisk denotes statistical significance (**, p < 0.01). NS denotes not significant.(H) Percentage of time in freezing in nicastrin cKO mice at 7–8 months of age in the contextual fear conditioning. Nicastrin cKO mice spend significantly less time in freezing than the controls 24 hours after the footshock (p < 0.00001; n = 13 for control; n = 10 for nicastrin cKO), but they exhibit comparable levels of freezing to control mice during the training period. The asterisk denotes statistical significance (****, p < 0.001).

(A) Left: Nissl stained sagittal brain sections of control mice at 6 and 9 months of age. Right: Nissl stained brain sections of nicastrin cKO mice. There is progressive loss of white and grey matters in the neocortex and hippocampus, and enlargement of lateral ventricles in 6- and 9-month-old nicastrin cKO mice.(B) Reduced MAP2 immunoreactivity in the neocortex and hippocampus of nicastrin cKO mice at 6 months of age.(C) Reduced MAP2 immunoreactivity in the neocortex and hippocampus of nicastrin cKO mice at 9 months of age.(D) Decreased synaptophysin immunoreactivity in the neocortex and hippocampus of nicastrin cKO mice at 6 months of age.(E) Decreased synaptophysin immunoreactivity in the neocortex and hippocampus of nicastrin cKO mice at 9 months of age.Scale bar:100 μm.

(A) Western analysis on protein levels of GFAP, a marker for astrocytes. There is a progressive increase of GFAP levels in cortical lysates of nicastrin cKO mice. At 2 months, GFAP levels are higher in nicastrin cKO mice (p < 0.05). However, at 6 months, there is an about 3-fold increase in GFAP levels in the cortex of nicastrin cKO mice.(B) Age-related increase of GFAP levels in PS cDKO mice. There is a small increase on GFAP levels between the control and PS cDKO mice at 2 months. At 6 months, there is a 2-fold increase in GFAP levels in PS cDKO mice.(C) Immunstaining of GFAP in the neocortex of nicastrin cKO mice. There is a progressive astrocytosis in nicastrin cKO mice. In nicastrin cKO mice, there is a small increase in GFAP immunoreactivity at 2 months and a dramatic increase at 6 months. Scale bar: 200 μm.(D) Western analysis on protein levels of Iba1, a marker for microglia. There is a progressive increase of Iba1 levels in cortical lysates of nicastrin cKO mice. At 2 months, Iba1 levels are higher in nicastrin cKO mice. At 6 months, there is a 6-fold increase in Iba1 protein levels. (E) Age-related increase of Iba1 levels in PS cDKO mice. There is a small increase in Iba1 levels in PS cDKO mice at 2 months, and a 3-fold increase in Iba1 levels at 6 months.(F) Immunstaining of Iba1 in the neocortex of nicastrin cKO mice. There is a progressive microgliosis in nicastrin cKO mice. Iba1 immunoreactivity is increased in the cKO cortex at 2 months, and highly elevated at 6 months. Scale bar: 100 μm.

(A) TUNEL staining in the neocortex of nicastrin cKO mice. TUNEL cells are shown in green. At 2 months, TUNEL cells can be seen in the cortex of nicastrin cKO mice. However, many more TUNEL cells can be found in nicastrin cKO mice at 6 months. Control mice do not show TUNEL cells. Scale bar: 25 μm.(B) Quantitative data on the average number of TUNEL cells per section. At 2 months, nicastrin cKO mice show more TUNEL cells in the cortex than control mice (control: n = 4; cKO: n = 5). At 6 months, there is a highly significant increase on the average number of TUNEL cells in nicastrin cKO mice (control: n = 4; cKO: n = 5).(C) Active caspase-3 immunostaining in the neocortex of nicastrin cKO mice. The sections were counter-stained with hematoxylin. Active caspase-3 positive (+) cells are shown in brown and non-active caspase-3 positive cells are shown in blue. At 2 months, active caspase-3 positive staining can be seen in nicastrin cKO mice. At 6 months, nicastrin cKO mice show many more cells with positive staining for active caspase-3 in the cortex. Scale bar: 25 μm.(D) Quantification of average numbers of active caspase-3 cells per section. At 2 months, the average number of active caspase-3 is higher in nicastrin cKO than in control mice (control: n = 4; cKO: n = 5). At 6 months, there is a large increase in the average number of active caspase-3 cells in nicastrin cKO mice (control: n = 4; cKO: n = 5).